Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes dengue fever (DF) and severe life threatening illness, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DENV is a small, enveloped, positive-stranded RNA virus that belongs to the Flavivirus genus of the Flaviviridae family. Four distinct subtypes or serotypes of dengue viruses (DV-1 to DV-4) are transmitted to humans through the bites of mosquito species Aedes aegypti and Aedes albopictus. It has been estimated that 50-100 million cases of DF and 250,000-500000 cases of DHF occur every year. Dengue constitutes a significant international public health concern, as two-fifths of the world's population live in dengue endemic regions, and an estimated 50-100 million cases of dengue infection occur annually. Furthermore 2.5 billion people are at risk for infection in subtropical and tropical regions of the world in the absence of effective intervention.
More than 100 tropical countries have endemic dengue virus infections, and DHF has been documented in >60 of these countries. Surveillance for DF/DHF is poor in most countries, and in the past has focused primarily on DHF; the number of DF cases that occur each year can therefore only be estimated. In 1998, however, major epidemics occurred throughout Asia and the Americas, with >1.2 million cases of DF/DHF reported to the World Health Organization (WHO). Global reports of DHF have increased on average by five-fold in the past 20 years. At the beginning of the 21st century it is estimated that between 50 and 100 million cases of DF and several hundred thousand cases of DHF occur each year, depending on the epidemic activity. The case fatality rate (CFR) varies among countries, but can be as high as 10-15% in some and <1% in others.
There are four dengue virus subtypes: dengue-1 (DV-1), dengue-2 (DV-2), dengue-3 (DV-3), and dengue-4 (DV-4). Each one of these subtypes form an antigenically distinct subgroup within the flavivirus family. They are enveloped, RNA viruses that encode ten proteins: three structural proteins and seven non-structural proteins. The structural proteins are capsid (C), envelope (E) and pre-membrane precursor (preM). The intracellular life cycle of DV begins with receptor-mediated endocytosis of the virus in to cells followed by fusion of the viral envelope protein with the late endosomal membrane, which results in the release of the viral genome into the cytoplasm for replication.
Infection by DV may either be asymptomatic or characterized by fever, chills, frontal headache, myalgia, arthralgia and rash. Subsequent infection with different serotypes may result in more severe manifestations of the disease involving plasma leakage or hemorrhage (dengue hemorrhagic fever) and shock (dengue shock syndrome). Although extensive studies have been carried out over the years to understand the pathogenicity of DENV infection, little progress has been made in the development of specific anti-DV compounds. Currently there are no specific anti viral agents or vaccines against Dengue infections approved in the US.
The envelope (E) glycosylated protein, being the major structural protein present on the surface of the mature dengue virions, is a type I integral membrane protein. It has been demonstrated that the E protein of the mature Dengue forms homodimers in the anti-parallel manner (head to tail orientation). Each monomer is folded into three distinct domains, namely domain I (DI, the central N-terminal domain), domain II (DII, the dimerization domain), and domain III (DIII, immunoglobulin (Ig) like C terminal domain). The DIII domain of E protein consists of 100 amino acids (residues 303-395) of the C-terminus. This domain has been suggested to be the receptor recognition and binding domain. Ig-like fold present in the DIII protein is commonly associated with structures that have an adhesion function. This domain extends perpendicularly to the surface of the virus, with a tip that projects further from the virion surface than any other part of the E protein. In addition, studies have demonstrated that both recombinant DIII proteins and antibodies generated against DIII of E protein of flavivirus can inhibit entry of the flavivirus into target cells. Further, flavivirus with mutation in DIII of the E protein shows either attenuated virulence or the ability to escape immune neutralization.
Development of a safe and effective vaccine against dengue virus infection remains a principal public health goal. Given that the primary correlate of immunity to dengue virus is thought to be the presence of neutralizing antibodies, a prerequisite for comparing and optimizing vaccine candidates is the ability to precisely measure the neutralizing antibody responses evoked by vaccines. A combination of live attenuated virus-containing vaccines from all four serotypes has been shown to result in several complications (Guy B, Almond J W, Comp Immunol Microbiol Infect Dis. 2008 March; 31(2-3):239-52). Further, there are few reports on an adenovirus-based delivery of dengue antigens. Nevertheless, the one well recognized problem with adenovirus systems is a majority of the human population is known to have antibodies against one of the adenoviruses, and such pre-existing antibodies can cause these adeno-based vaccines to be ineffective.
Therefore, there remains a need to develop a vaccine that provides broad immunity against multiple and preferably all four serotypes of dengue virus, or universal immunity, and preferably a vaccine which is economical and effective across all serotypes. Further, there remains a need for an effective method of administering vaccines, such as DNA vaccines or DNA plasmid vaccines, to a mammal in order to provide immunization against dengue virus, either prophylatically or therapeutically.